Parachlorella polysaccharide, and preparation method and application thereof

Abstract

The present application belongs to the technical field of biological medicine, and relates to a kind of Parachlorella sp. Polysaccharide and its preparation method and application. The present application obtains a kind of uniform Parachlorella sp. Polysaccharide NGP-1 by water extraction, alcohol precipitation, column chromatography separation to Parachlorella sp.;Combining high performance liquid chromatography, methylation combined gas chromatography-mass spectrometry, nuclear magnetic resonance and other methods to determine its structural characteristics. The obtained NGP-1 can promote macrophage proliferation, enhance phagocytic ability, improve active oxygen level, induce nitric oxide and cytokine production, and up-regulate the expression of corresponding mRNA, and has significant immune enhancing activity. The Parachlorella sp. Polysaccharide NGP-1 prepared by the present application can be used as a natural immune enhancer, which widens the application range of Parachlorella sp. And has broad application prospect.

Description

[Technical Field]

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polysaccharide of *Micrococcus pseudocarpa*, its preparation method, and its application. [Background Technology]

[0002] Polysaccharides are macromolecular compounds widely found in plants, animals, and microorganisms. Due to their unique structure and function, they have significant applications in the biomedical field, especially in immunomodulation. In recent years, with the deepening research on algal polysaccharides, many algal polysaccharides have been found to possess significant immunomodulatory activities. However, the composition and structural characteristics of microalgal polysaccharides vary significantly among different species, exhibiting obvious species dependence. Currently, various novel and regularly structured microalgal polysaccharides are being continuously discovered from different microalgae.

[0003] *Micrococcus pseudochlorella*, also known as *Micrococcus pseudochlorella*, is a single-celled microalga. Current research on the utilization of *Micrococcus pseudochlorella* resources focuses primarily on increasing lipid accumulation, specifically by adjusting culture conditions such as nitrogen and carbon sources to increase lipid production, which can then be used for the production of eicosapentaenoic acid (EPA) and biofuels (Waste and Biomass Valorization, 2019, 10.1007 / s12649-018-00552-2; Journal of Renewable and Sustainable Energy, 2013, 5, 063138; Nature Biotechnology, 2017, 10.1038 / nbt.3865). In terms of bioactivity, studies have shown that *Nannochloropsis gaditana* possesses hepatoprotective, anti-inflammatory, antioxidant, and skin-protective effects (Nutrients, 2023, 15, 1960; Pharmacological Research, 2018, 128, 220-230; Marine Drugs, 2022, 20, 318; Frontiers in Marine Science, 2017, 4, 221). *Nannochloropsis gaditana* is rich in nutrients such as protein and eicosapentaenoic acid (EPA). In 2021, the National Health Commission of China issued the "Announcement on Six New Foods Including β-1,3 / α-1,3-glucan" (No. 5 of 2021), in which *Nannochloropsis gaditana* was one of the approved new food ingredients, demonstrating broad development and application prospects. Although *Nannochloropsis gaditana* shows great potential in resource utilization and bioactivity, current research still has some shortcomings. Current research mainly focuses on lipids or proteins, and some studies even lack clear classification of its extracts. Studies on the structure and activity of *Microcystis aeruginosa* polysaccharides are rarely reported. Therefore, in-depth research on the structure and function of *Microcystis aeruginosa* polysaccharides will help to further explore their application potential in the fields of medicine and health care. [Summary of the Invention]

[0004] The purpose of this invention is to provide a polysaccharide from *Micrococcus pseudocarpa*, its preparation method, and its applications. The applicant extracted and purified a polysaccharide with a well-defined structure from *Micrococcus pseudocarpa*. Activity studies have shown that this polysaccharide possesses significant immunomodulatory effects and has broad application prospects in the development of novel immunomodulators.

[0005] To achieve the above objectives, the present invention provides a *Micrococcus pluvialis* polysaccharide, the structural formula of which is shown in formula (I):

[0006]

[0007] In equation (I), m is an integer ranging from (57±5) to (28±5); n is an integer ranging from (3±2);

[0008] The weight-average molecular weight of the *Micrococcus pluvialis* polysaccharide is 5k to 10kDa, and the polydispersity index is 1.0 to 1.8.

[0009] To further explain, the polysaccharide of *Micrococcus pseudospp.* is composed of glucuronic acid, mannose, and glucose, wherein the molar percentages of glucuronic acid, mannose, and glucose are (1.48±0.5)%, (1.90±0.50)%, and (96.62±10.00)%, respectively.

[0010] Further explanation: the *Microcystis globulus* polysaccharide includes sugar residues such as β-Glcp(1→, →3)-β-Glcp-(1→, →4)-β-Glcp-(1→, →4)-β-Glcp and →4)-α-Glcp, wherein β-Glcp-(1→ accounts for (6.2±2.0)%; →4)-β-Glcp-(1→ accounts for (88.3±5.0)%; and →3)-β-Glcp-(1→ accounts for (4.1±2.0)%.

[0011] The present invention also provides a method for preparing the above-mentioned microalgae polysaccharide, comprising the following steps:

[0012] (1) Preparation of crude polysaccharide: Take *Micrococcus pseudochlorella* powder or defatted *Micrococcus pseudochlorella* powder and extract it in hot water at 80-100℃ for 2-4 hours at a ratio of 1:20-40 g / mL. Repeat the extraction 1-3 times, combine the supernatants, add 3-4 times or more of 95% ethanol or anhydrous ethanol to precipitate, redissolve the precipitate, and repeatedly remove the protein 2-6 times using the Sevag method. Then freeze-dry to obtain crude polysaccharide of *Micrococcus pseudochlorella*.

[0013] (2) Isolation and purification of polysaccharides: Crude polysaccharides were purified by anion exchange column. After loading the crude polysaccharides, gradient elution was performed. The eluents were sodium chloride solutions of different concentrations: 0M, 0.1M, 0.3M, and 0.5M. The eluted fractions of 0M and 0.1M sodium chloride solutions were collected, and the polysaccharides were desalted by dialysis, gel column chromatography or ultrafiltration membrane treatment, and then lyophilized to obtain the polysaccharides of *Micrococcus pseudocarpa*.

[0014] This invention also provides an application of the above-mentioned microcystin polysaccharide in the preparation of an immune enhancer, which enhances macrophage immune activity by activating TLR4 / TLR2 receptors, upregulating the phosphorylation levels of key proteins in the MAPK and NF-κB pathways.

[0015] To further explain, the enhancement of macrophage immune activity includes promoting macrophage proliferation, enhancing phagocytic capacity, increasing ROS levels, inducing the secretion of nitric oxide and cytokines TNF-α, IL-6, and IL-1β, and upregulating the corresponding mRNAs.

[0016] The present invention also provides a pharmaceutical composition comprising the above-described microsporum polysaccharide, comprising an effective amount of microsporum polysaccharide.

[0017] Furthermore, the pharmaceutical composition also contains a pharmaceutically acceptable carrier or excipient.

[0018] Typically, pharmaceuticals are clinically used only after being formulated into a pharmaceutical composition. The pharmaceutical compositions described in this invention can be prepared according to methods known in the art. They can be formulated into any dosage form suitable for human or animal use by combining the pharmaceutical compositions of this invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants.

[0019] The pharmaceutical composition of the present invention, or a pharmaceutical composition containing it, can be administered in unit dose form, and the route of administration can be enteric or non-enteric, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0020] Dosage forms can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc.

[0021] The pharmaceutical compositions of the present invention can be formulated into conventional formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various particulate delivery systems. To formulate the pharmaceutical compositions of the present invention into tablets, a wide range of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, croscarmellose, croscarmellose sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0022] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0023] To formulate the drug delivery unit into capsules, the active ingredient, the pharmaceutical composition of the present invention, can be mixed with a diluent and a disintegrant, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient, the pharmaceutical composition of the present invention, can be first formed into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. The diluents, binders, wetting agents, disintegrants, and disintegrants used to prepare tablets of the pharmaceutical composition of the present invention can also be used to prepare capsules of the pharmaceutical composition of the present invention.

[0024] To prepare the pharmaceutical composition of the present invention into an injection, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, co-solvents, pH adjusters, and osmotic pressure regulators can be added. Solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure regulators can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing a lyophilized powder injection, mannitol, glucose, etc., can also be added as a support agent.

[0025] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0026] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0027] (1) The present invention obtains polysaccharide (NGP-1) from *Nyctaginella spp.* through hot water extraction, ethanol precipitation, protein removal, and anion exchange column chromatography. The preparation process is simple and easy to operate.

[0028] (2) The NGP-1 obtained in this invention is derived from the inexpensive and abundant micrococcus. It has a low molecular weight, a simple and regular structure, and a clear and well-defined composition.

[0029] (3) Experiments show that the NGP-1 obtained in this invention can promote macrophage proliferation, enhance phagocytic function, increase ROS levels, and significantly induce the secretion of NO, TNF-α, IL-6, and IL-1β. Its mechanism of action is clear: by activating TLR4 / TLR2 receptors, it upregulates the phosphorylation levels of key proteins in MAPKs (Erk1 / 2, p38, JNK) and the NF-κB pathway, thereby achieving an immune-enhancing effect. Within a concentration range of 12.5 μg / mL to 200 μg / mL, NGP-1 did not exhibit cytotoxicity, indicating its safety in clinical applications. NGP-1 can be used as a novel immune enhancer to treat immunodeficiency diseases (such as infection, immunosuppression after radiotherapy and chemotherapy for tumors), or as a functional food additive to enhance the body's immunity. Furthermore, its low molecular weight and structural stability are beneficial for drug formulation development (such as oral tablets and injections), showing promising application prospects. [Attached Image Description]

[0030] Figure 1 This is a high-performance liquid chromatography gel permeation chromatogram showing the molecular weight distribution of the microalgae polysaccharide (NGP-1) of the present invention.

[0031] Figure 2 This is a high-performance liquid chromatogram of the monosaccharide composition analysis of the *NGP-1* polysaccharide of the present invention.

[0032] Figure 3 This is the total ion chromatogram of the methylated sugar alcohol acetyl derivative of the microalgae polysaccharide (NGP-1) of the present invention.

[0033] Figure 4 This is the hydrogen spectrum of the microalgae polysaccharide (NGP-1) of the present invention.

[0034] Figure 5 This is the carbon spectrum of the microalgae polysaccharide (NGP-1) of the present invention.

[0035] Figure 6 The present invention is a polysaccharide of *N. chrysophagus* (NGP-1). 1 H- 1 H COSY spectrum.

[0036] Figure 7The present invention is a polysaccharide of *N. chrysophagus* (NGP-1). 1 H- 1 H TOCSY spectrum.

[0037] Figure 8 The present invention is a polysaccharide of *N. chrysophagus* (NGP-1). 1 H- 13 C HSQC spectrum.

[0038] Figure 9 The present invention is a polysaccharide of *N. chrysophagus* (NGP-1). 1 H- 1 H ROESY spectrum.

[0039] Figure 10 The present invention is a polysaccharide of *N. chrysophagus* (NGP-1). 1 H- 13 C HMBC spectrum.

[0040] Figure 11 The effect of the microalgae polysaccharide (NGP-1) of the present invention on the proliferation ability of RAW264.7 cells.

[0041] Figure 12 The effects of the microalgae polysaccharide (NGP-1) of the present invention on NO (A) release and TNF-α (B), IL-6 (C), and IL-1β (D) secretion in RAW264.7 cells.

[0042] Figure 13 The effects of the microalgae polysaccharide (NGP-1) of the present invention on the expression levels of iNOS (A), TNF-α (B), IL-6 (C), and IL-1β (D) mRNA in RAW264.7 cells.

[0043] Figure 14 The effect of the microalgae polysaccharide (NGP-1) of the present invention on the phagocytic ability of RAW264.7 cells.

[0044] Figure 15 The effect of the microalgae polysaccharide (NGP-1) of the present invention on the ROS level of RAW264.7 cells.

[0045] Figure 16 Effects of TLR4 / TLR2 receptor inhibitor treatment on the promotion of NO (A), TNF-α (B), and IL-6 (C) production in RAW264.7 cells by the polysaccharide NGP-1 of the present invention.

[0046] Figure 17This invention illustrates the effect of *Nyctaginus polysaccharide-1* on the phosphorylation levels of key proteins in the NF-κB pathway in RAW264.7 cells. Western blotting was used to detect key proteins in the NF-κB pathway (A), and the results of relative quantification analysis are shown in (B).

[0047] Figure 18 This invention relates to the effect of *Nyctaginus polysaccharide-1* on the phosphorylation levels of key proteins in the MAPK pathway in RAW264.7 cells. Western blotting was used to detect key proteins in the MAPK pathway (A), and relative quantification results are shown in (B).

Detailed Implementation Methods

[0048] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0049] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0050] Example 1: Preparation and structural analysis of a microalgae polysaccharide NGP-1

[0051] 1. Extraction and purification of polysaccharides from *Microsporum pseudochlorium*

[0052] Take 30g of defatted *Nannochloropsis gaditana* algal powder and place it in an Erlenmeyer flask. Add 600mL of pure water at a material-to-liquid ratio of 1:20 (w / v) to disperse the powder, and extract in a water bath preheated to 90℃. After 3 hours, cool the solution to room temperature, centrifuge at 4700rpm for 15 minutes, and collect the supernatant. Add pure water to the filter residue and extract again at 90℃. After extraction for 3 hours, cool and centrifuge (4700rpm × 15min) to collect the supernatant. Combine the supernatants from the two extractions, add anhydrous ethanol under stirring, and add an appropriate amount of saturated sodium chloride. Refrigerate and let stand for 24 hours for salting-out alcohol precipitation. Centrifuge at 4700rpm for 15 minutes to collect the precipitate and dissolve it in an appropriate amount of pure water. Remove the protein from the polysaccharide solution using the Sevag method, repeating 4 times. Perform salting-out alcohol precipitation on the remaining sample. Finally, the precipitate was collected by centrifugation at 4700 rpm for 15 min, dissolved in pure water, concentrated, and freeze-dried to obtain crude polysaccharide from *Micrococcus pseudocarpa*.

[0053] 2. Isolation and purification of *Micrococcus pseudocarpa* polysaccharides

[0054] Weigh 500 mg of the crude polysaccharide from the previous step and dissolve it in an appropriate amount of pure water. Purify the polysaccharide using a DEAE Sepharose Fast Flow anion exchange column (45 cm × 3 cm). Gradient elution was performed with NaCl solutions of 0 M, 0.1 M, 0.3 M, and 0.5 M as eluents at a flow rate of 2 mL / min. The eluent was collected using an automated collector, divided into 10 mL tubes. Elution curves were monitored using the phenol-sulfuric acid method. The elution peaks from the 0.1 M sodium chloride solution were combined based on the elution curves, concentrated, dialyzed for desalting, concentrated again, and lyophilized to obtain the polysaccharide of this invention, named NGP-1.

[0055] 3. Determination of the molecular weight and distribution of the microalgae polysaccharide NGP-1 of the present invention.

[0056] NGP-1 polysaccharide solutions were prepared, and the molecular weight and distribution of NGP-1 were determined by high-performance gel permeation chromatography (HPGPC) using dextran of different molecular weights (2700, 5250, 9750, 13050, 36800 Da) as standards. Chromatographic analysis conditions: A Shimadzu LC-2030C 3D HPLC system was used; a Shodex OHpak SB-804HQ column (8×300 mm) was used at 35℃; the mobile phase was 0.1 M NaCl solution, and the flow rate was controlled at 0.5 mL / min; a differential refractive index detector was used.

[0057] 4. Monosaccharide composition analysis of the microalgae polysaccharide NGP-1 of the present invention

[0058] The monosaccharide composition of NGP-1 was determined by pre-column derivatization combined with high-performance liquid chromatography (HPLC) using PMP. The chromatographic conditions were as follows: the HPLC system was a Shimadzu LC-2030C 3D HPLC; the column was an Eclipse Plus C18 (4.6 × 250 mm, 5 μm), and the column temperature was 30 °C; the mobile phase was a mixture of phosphate buffer (0.1 M, pH 6.7) and acetonitrile at a volume ratio of 83:17, the flow rate was 1.0 mL / min, and the injection volume was 20 μL; the detector was a photodiode array detector (PDA) with a detection wavelength of 245 nm.

[0059] 5. Analysis of the glycosidic bond linkage mode of the microalgae polysaccharide NGP-1 of the present invention

[0060] The glycosidic bond linkage of NGP-1 was analyzed using a methylation-GC-MS method. 5 mg of the polysaccharide sample NGP-1 was placed in a round-bottom flask, and 2 mL of anhydrous DMSO was added. The mixture was magnetically stirred until completely dissolved. Approximately 50 mg of NaOH was added and stirred until dissolved. The flask was then placed in an ice bath to cool until the solution became viscous. 1.5 mL of CH3I was added, and the reaction was carried out in the dark for 2 hours. The reaction was terminated by adding 2 mL of pure water to the reaction solution. The methylated polysaccharide was extracted and dried. The above steps were repeated to ensure complete methylation of the polysaccharide.

[0061] Take the methylated sample, add 3 mL of 2MTFA solution, hydrolyze at 120℃ for 2 h, evaporate to dryness using a rotary evaporator, add 3 mL of methanol, and evaporate to dryness under reduced pressure again. Repeat 3 times. Add 2 mL of pure water to dissolve the sample, adjust the pH to alkaline, add 30 mg of NaBH4 for reduction for 3 h, and add glacial acetic acid dropwise to remove excess NaBH4. Repeatedly add methanol and concentrate to dryness under reduced pressure. Add 1.5 mL of acetic anhydride and 1.5 mL of pyridine to the dried residue, react at 100℃ for 1 h, add 1.5 mL of water to the reaction solution, then add 3 mL of dichloromethane for extraction, wash 3 times with 2 mL of pure water, and evaporate to dryness to obtain the methylated sugar alcohol acetyl derivative. Dissolve in 100 μL of dichloromethane and perform gas chromatography-mass spectrometry (GC-MS) analysis.

[0062] Test conditions: Agilent 7890B-5977B gas chromatograph-mass spectrometer; capillary column: HP-5ms (30m×0.25mm×0.25μm); carrier gas: He; temperature program: 50℃ for 1 min, 50-130℃ (50℃ / min), 130-230℃ (3℃ / min) for 2 min; ionization mode: EI (70kV).

[0063] 6. Nuclear magnetic resonance spectroscopy analysis

[0064] Weigh 30 mg of dried NGP-1 and dissolve it in 0.6 mL of D2O. Centrifuge and collect the supernatant. Freeze-dry the supernatant, repeating this process three times. Dissolve the freeze-dried sample in 0.5 mL of D2O containing the internal standard, and determine the 1D and 2D NMR spectra as shown below. 1 H, 13 C. COSY, TOCSY, ROESY, HSQC, HMBC.

[0065] 7. Experimental Results

[0066] (1) Molecular weight and its distribution

[0067] The high-performance liquid chromatography gel permeation chromatogram of NGP-1 is attached. Figure 1 As shown, NGP-1 appears as a single peak in the HPGPC chromatogram, indicating high purity; based on the standard curve equation, the molecular weight of NGP-1 is calculated to be 8087 Da.

[0068] (2) Monosaccharide composition

[0069] The monosaccharide composition of NGP-1 is shown in the attached figure. Figure 2 As shown, a comparison with monosaccharide standards reveals that the monosaccharide composition of NGP-1 is mainly glucose, with small amounts of mannose and glucuronic acid, and the molar percentages are 96.62%, 1.90%, and 1.48%, respectively.

[0070] (3) Methylation analysis

[0071] The total ion chromatogram of NGP-1 methylated sugar alcohol acetyl derivatives is attached. Figure 3 As shown. Methylation analysis results indicate that NGP-1 has five glycosidic bond types, with →4)-Glcp-(1→ being the predominant type. The molar percentages of each bond type are as follows: →4)-Glcp-(1→ (88.28%), Glcp-(1→ (6.24%), →3)-Glcp-(1→ (4.10%), →4)-Manp-(1→ (1.38%), and →3,4)-Glcp-(1→ (1.36%).

[0072] (4) Nuclear magnetic resonance spectroscopy analysis

[0073] The structure of NGP-1 was further characterized using nuclear magnetic resonance spectroscopy techniques (1D NMR and 2D NMR). 1 H, 13 C, COSY, TOCSY, HSQC, ROESY, HMBC, etc. are shown in the attached spectrum. Figures 4 to 10 As shown in Table 1, the chemical signals attributing hydrogen and carbon to each sugar residue were determined based on the NMR spectra.

[0074] Table 1. NGP-1 1 H and 13 C NMR signal attribution

[0075]

[0076] Note: Bold text indicates connection sites.

[0077] The polysaccharide NGP-1 from *Micrococcus pluvialis* was characterized by monosaccharide composition, methylation analysis, and nuclear magnetic resonance spectroscopy, and its structural formula is inferred as follows:

[0078]

[0079] NGP-1 is a low molecular weight dextran whose sugar chain starts from the non-reducing end β-Glcp-(1→, mainly containing →4)-β-Glcp-(1→, with a small amount of →3)-β-Glcp-(1→, and the reducing end is →4)-β-Glcp or →4)-α-Glcp, where the mean m is 46 and the mean n is 2.

[0080] Example 2: Immunomodulatory activity of NGP-1, a polysaccharide from *Micrococcus pluvialis* prepared in this invention.

[0081] 1. Cell Culture

[0082] 10% fetal bovine serum and 1% penicillin antibiotics were added to DMEM medium to make complete culture medium for RAW264.7 cells. The cells were cultured in an incubator with 5% CO2 and 37°C.

[0083] 2. Effect of the *N. GP-1* polysaccharide prepared in this invention on the proliferation ability of RAW264.7 cells.

[0084] The effect of NGP-1 on the proliferation of RAW264.7 cells was detected using the MTT assay. Cells in the logarithmic growth phase were collected and the cell density was adjusted to 8 × 10⁻⁶ cells / year. 4 Cells were seeded and treated using 96-well plates, 100 μL per well. Cells were incubated overnight at 37°C in a 5% CO2 incubator, and the supernatant was discarded. 100 μL of DMEM complete medium, different concentrations (12.5, 25, 50, 100, 200 μg / mL) of NGP-1 polysaccharide solution, or LPS solution (1 μg / mL, prepared with DMEM complete medium) were added to each well, with 3 to 5 replicates per group. After 24 h of incubation, the supernatant was discarded, and 100 μL of 0.5 mg / mL MTT solution was added to each well. Cells were incubated at 37°C in a 5% CO2 incubator for 3–4 h, and the supernatant was discarded. 150 μL of LDMSO (analytical grade) was added to each well, and the plates were gently shaken to dissolve any crystals. Cell viability was calculated by measuring the absorbance at 490 nm using a microplate reader.

[0085] 3. Effect of the *N. 1* polysaccharide NGP-1 prepared in this invention on NO release in RAW264.7 cells.

[0086] Standard curve plotting: Dilute 1 mmol / L NaNO2 standard solution to a series of gradient concentrations (1, 2, 5, 10, 20, 40, 60, 100 μmol / L) with DMEM complete medium. Pipette 50 μL of each concentration of NaNO2 standard solution into a 96-well plate, then add equal volumes of Griess A and Griess B solutions sequentially, and incubate in the dark for 10 min. Measure the absorbance at 540 nm using a microplate reader to plot the standard curve.

[0087] Collect cells in the logarithmic growth phase and adjust the cell density to 4 × 10⁻⁶. 5 Cells were treated as described above, and 24 hours after drug administration, the supernatant was collected. Equal volumes of Griess A and Griess B solutions were added sequentially, and the reaction was carried out in the dark for ten minutes. The absorbance at 540 nm was measured using a microplate reader, and the NO release was determined using a standard curve.

[0088] 4. Effect of the *N. 1* polysaccharide prepared in this invention on the secretion of cytokines in RAW264.7 cells.

[0089] Cells were seeded and treated according to the method for detecting NO, and the supernatant was collected 24 hours after drug administration. The levels of TNF-α, IL-6, and IL-1β in the cell supernatant were detected according to the ELISA kit instructions.

[0090] 5. Effects of the *Nyctaginus polysaccharide* NGP-1 prepared in this invention on the expression of iNOS, TNF-α, IL-6, and IL-1β mRNA in RAW264.7 cells.

[0091] Take cells in the logarithmic growth phase and adjust the cell density to 4 × 10⁻⁶. 5 Cells were seeded at 2 mL / well in 6-well plates and incubated overnight at 37°C in a 5% CO2 incubator. The supernatant was discarded. Cells were treated as described above, and the supernatant was discarded 24 h after drug administration. After treatment with PBS, total RNA was extracted from the cells using TRIzol reagent. The extracted total RNA was transcribed into cDNA, which was then used as a template for subsequent amplification reactions. The primer sequences used in this example are shown in Table 2. The amplification program was set as follows: 95°C for 30 s pre-denaturation; 95°C for 6 s denaturation, 60°C for 30 s annealing and extension, 40 cycles; 40°C for 10 s. GAPDH was used as an internal control. -△△Ct The method is used to quantitatively analyze the target gene in the sample.

[0092] Table 2 Gene Primer Sequences

[0093]

[0094] 6. Effect of the *N. phagocytic ability of* RAW264.7 cells on the polysaccharide NGP-1 prepared in this invention.

[0095] Cells were seeded and treated using 48-well plates at a density of 2 × 10⁶ cells / well. 5Cells / mL. After treating cells with the above method for 24 h, 5 μL of FITC-dextran stock solution (40,000 Da, 20 mg / mL) was added to each well to a final concentration of 1 mg / mL, and the cells were incubated at 37°C in the dark for 1 h. The culture medium was discarded, the cells were washed with PBS and collected, and the fluorescence intensity of each group of cells was detected by flow cytometry to assess the phagocytic capacity of the cells.

[0096] 7. Effect of the *N. 1* polysaccharide prepared in this invention on ROS levels in RAW264.7 cells.

[0097] ROS levels in RAW264.7 cells were detected using the DCFH-DA probe. Cells were seeded and treated using the same method as in phagocytosis experiments. After 24 h of treatment, cells were washed twice with PBS. 100 μL of the prepared Photo-oxidation Resistant DCFH-DA working solution was added to each well, and the cells were incubated at 37 °C for 30 min in a 5% CO2 incubator. The working solution was removed, cells were washed with PBS, and fluorescence intensity was detected by flow cytometry.

[0098] 8. Detection of the interaction receptors between the polysaccharide NGP-1 prepared in this invention and RAW264.7 cells.

[0099] Cells were seeded and treated using 96-well plates at a density of 4 × 10⁶ cells / well. 5 Cells were pretreated with TLR4 inhibitor (TAK-242, 1 μM) and TLR2 inhibitor (C29, 100 μM) for 1 h, respectively. Cells were then incubated with NGP-1 (200 μg / mL) for 24 h. The supernatant was used to measure NO, TNF-α, and IL-6.

[0100] 9. The activation effect of the *N. 264.7* MAPKs and NF-κB signaling pathways on *N. 264.7* polysaccharide NGP-1 prepared in this invention.

[0101] Cells were seeded and treated using 12-well plates at a density of 1 × 10⁶ cells / well. 6Cells were injected into each well at 1.2 mL / well. The following groups were established: blank control group (DMEM medium containing 10% FBS), LPS group (1 μg / mL), and NGP-1 group (50, 100, and 200 μg / mL). Two hours after administration, the supernatant was discarded, and the cells were washed twice with PBS. Cells were lysed with lysis buffer, and total protein was extracted and quantified using a BCA kit. Protein samples were separated by 12.5% ​​SDS-PAGE and then electroporated onto 0.45 μm PVDF membranes. The membranes were blocked with 5× protein-free rapid blocking buffer for 10 min, followed by incubation with primary antibody (MAPK Erk1 / 2, p-Erk1 / 2, p38, p-p38, JNK, p-JNK, NF-κBp65, p-NF-κBp65, IκB-α, p-IκB-α, and β-actin) at 4°C overnight. After washing with TBST, the membranes were incubated with secondary antibody for 1 h. Protein bands via Omni-ECL TM A high-efficiency chemiluminescence assay kit was used for color development, and the results were detected using a chemiluminescence image analyzer. Finally, ImageJ software was used for relative quantification of protein expression levels.

[0102] 10. Results

[0103] (1) Effect of the polysaccharide NGP-1 prepared in this invention on the proliferation ability of RAW264.7 cells.

[0104] As attached Figure 11 As shown, compared with the blank control group, NGP-1 showed no cytotoxicity in the concentration range of 12.5 μg / mL to 200 μg / mL and could promote cell proliferation, which can be used for subsequent experimental determination.

[0105] (2) Effects of the microcystis polysaccharide NGP-1 prepared in this invention on NO release and cytokine secretion in RAW264.7 cells

[0106] As attached Figure 12 As shown, compared with the blank control group, NGP-1 significantly promoted the release of NO and the secretion of pro-inflammatory factors TNF-α, IL-6 and IL-1β in RAW264.7 cells in the concentration range of 12.5 μg / mL to 200 μg / mL (P<0.05), and all showed good concentration dependence.

[0107] (3) Effects of the polysaccharide NGP-1 prepared in this invention on the expression of iNOS, IL-6, IL-1β and TNF-α mRNA in RAW264.7 cells

[0108] As attached Figure 13As shown, after 24 hours of NGP-1 treatment, the expression levels of iNOS, IL-6, IL-1β, and TNF-α mRNA in RAW264.7 cells were upregulated in a concentration-dependent manner. This indicates that NGP-1 activates RAW264.7 cells at the mRNA level, exerting its immune-enhancing effect.

[0109] (4) Effect of the microcystis polysaccharide NGP-1 prepared in this invention on the phagocytic ability of RAW264.7 cells.

[0110] As attached Figure 14 As shown, compared with the blank control group, the mean fluorescence intensity (MFI) of RAW264.7 cells was significantly enhanced after treatment with NGP-1 at concentrations ranging from 12.5 μg / mL to 200 μg / mL (P<0.05). This indicates that NGP-1 can activate macrophages and enhance their phagocytic function in a dose-dependent manner.

[0111] (5) Effect of the polysaccharide NGP-1 prepared in this invention on ROS levels in RAW264.7 cells

[0112] Reactive oxygen species (ROS) are crucial for macrophages to clear invading microorganisms and also play a key role in non-pathogen killing processes (such as signal transduction, cell differentiation, and gene expression regulation) (Frontiers in Immunology, 2021, 12, 734229). To investigate whether NGP-1 can induce ROS production in RAW264.7 macrophages, RAW264.7 cells were incubated with NGP-1 (200 μg / mL) or LPS (1 μg / mL) for 24 h. The results are attached. Figure 15 As shown, at a concentration of 50 μg / mL, cellular ROS production significantly increased, exhibiting a concentration-dependent effect within the range of 50 μg / mL to 200 μg / mL. This indicates that NGP-1 treatment mediates the upregulation of intracellular ROS production.

[0113] (6) Effect of TLR2 / TLR4 inhibitor blockade on the immuno-enhancing effect of NGP-1 polysaccharide prepared in this invention

[0114] As attached Figure 16 As shown, compared with NGP-1 treatment alone, treatment with TLR2 and TLR4 receptor inhibitors significantly reduced NO release, TNF-α, and IL-6 secretion in RAW264.7 cells, indicating that TLR2 and TLR4 are key receptors for the interaction between NGP-1 and RAW264.7 cells. Furthermore, compared with C29, TAK242 treatment resulted in a more significant decrease in the immune activity of RAW264.7 cells, suggesting that NGP-1-induced macrophage activation is primarily mediated through the TLR4 receptor pathway.

[0115] (7) Effects of the polysaccharide NGP-1 prepared in this invention on phosphorylation of key proteins in the MAPK and NF-κB pathways in RAW264.7 cells.

[0116] As attached Figure 17 , 18 As shown, after 2 hours of NGP-1 treatment, the phosphorylation levels of key proteins in the MAPK and NF-κB pathways in RAW264.7 cells were significantly increased (P<0.05) in a dose-dependent manner. This indicates that NGP-1 can activate macrophages and exert its immune-enhancing effect by increasing the phosphorylation levels of key proteins in the MAPK and NF-κB pathways.

[0117] In summary, the *NGP-1* polysaccharide prepared by this invention is simple to prepare, has a well-defined structure, exhibits significant immunomodulatory activity, and is non-cytotoxic. It is a promising candidate compound for development as an immunomodulator and has excellent application prospects.

[0118] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A polysaccharide derived from *Micrococcus pluvialis*, characterized in that: The structural formula of the pseudomicrophyllum polysaccharide is as shown in formula (I): （I）； In equation (I), m is an integer from (57±5) to (28±5); n is an integer from (3±2); The weight-average molecular weight of the *Micrococcus pluvialis* polysaccharide is 5k to 10kDa, and the polydispersity index is 1.0 to 1.

8. The *Micrococcus pseudochlorella* polysaccharide is composed of glucuronic acid, mannose, and glucose, wherein the molar percentages of glucuronic acid, mannose, and glucose are (1.48±0.5)%, (1.90±0.50)%, and (96.62±10.00)%, respectively. The microsporin polysaccharide includes β-Glc p (1→、→3)-β-Glc p -(1→、→4)-β-Glc p -(1→、→4)-β-Glc p →4)-α-Glc p Isoglycolic residues, of which β-Glc p -(1→accounts for (6.2±2.0)%;→4)-β-Glc p -(1→accounts for (88.3±5.0)%;→3)-β-Glc p -(1→accounts for (4.1±2.0)%).

2. The method for preparing *Micrococcus pseudocarpa* polysaccharide as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of crude polysaccharide: Take *Micrococcus pseudochlorella* powder or defatted *Micrococcus pseudochlorella* powder, extract it in hot water at 80-100℃ for 2-4 hours at a ratio of 1:20-40 g / mL, repeat 1-3 times, combine the supernatant, add 3-4 times or more of 95% ethanol or anhydrous ethanol to precipitate, after redissolve the precipitate, repeatedly remove protein 2-6 times by the Sevag method, and freeze dry to obtain crude polysaccharide of *Micrococcus pseudochlorella*. (2) Isolation and purification of polysaccharides: Crude polysaccharides were purified by anion exchange column. After loading the crude polysaccharides, gradient elution was performed. The eluents were sodium chloride solutions of different concentrations: 0M, 0.1M, 0.3M, and 0.5M. The eluted fractions of 0M and 0.1M sodium chloride solutions were collected, and the polysaccharides were desalted by dialysis, gel column chromatography or ultrafiltration membrane treatment, and then lyophilized to obtain the polysaccharides of *Micrococcus pseudocarpa*.

3. The application of the microcystin polysaccharide as described in claim 1 in the preparation of immune enhancers enhances macrophage immune activity by activating TLR4 / TLR2 receptors, upregulating the phosphorylation levels of key proteins in the MAPK and NF-κB pathways.

4. The application as described in claim 3, characterized in that: The enhancement of macrophage immune activity includes promoting macrophage proliferation, enhancing phagocytic capacity, increasing ROS levels, inducing the secretion of nitric oxide and cytokines TNF-α, IL-6, and IL-1β, and upregulating the corresponding mRNAs.

5. A pharmaceutical composition comprising the polysaccharide of *Micrococcus pseudocarpa* according to claim 1, characterized in that: It contains an effective amount of microalgae polysaccharides.

6. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

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